Botulinum toxin is a neurotoxin produced by the Gram-positive anaerobic bacterium Clostridium botulinum, which grows in spoiled canned goods or spoiled meat. Botulinum toxin is classified into 8 types of neurotoxins, seven types (A, B, C, D, E, F, G) of which may induce neuronal paralysis. Botulinum toxin has a size of approximately 150 kDa, and forms a complex of a botulinum toxin protein and a non-toxin protein. The size of each complex is formed to have a size up to 900 kDa according to the type of neurotoxin. Action type and target, and an activity duration may vary according to botulinum toxin type, and the botulinum toxin type A is known as one of the deadly biological agents.
Botulinum toxin causes paralysis by blocking a signal inducing muscle convulsion or contraction, and due to this function, is used for medical treatment or cosmetic purposes, since approved by the FDA in 1989. For medical treatment, botulinum toxin is used as an injection for a medical purpose to treat a neuromuscular disorder such as strabismus, torticollis or blepharospasm, for a cosmetological purpose to reduce wrinkles, frown or glabellar lines and a square jaw, and for other purposes to treat hyperhidrosis or migraines. While it has been reported that botulinum toxin has side effects including dysphagia, voice change, dry mouth and blurred vision, since no death directly caused by botulinum toxin has been reported yet, if properly used, botulinum toxin is evaluated as a very safe drug. However, the use of botulinum toxin is restricted in the cases of a person who has hypersensitivity to the drug or a musculoskeletal disease, or a pregnant or breastfeeding woman.
In current applications of botulinum toxin, the duration of botulinum toxin injected into skin tissue lasts approximately 3 to 6 months, and when signal transduction between a nerve and a muscle is blocked by botulinum toxin, a new dendrite is produced to reduce a neuronal paralysis effect caused by botulinum toxin, and thus a regular treatment is needed. Also, when botulinum toxin is repeatedly administered, an antibody to the botulinum toxin is produced in vivo, and thus its effect is reduced.
Also, since muscle paralysis caused by such botulinum toxin is mostly induced by injections, a variety of research has been conducted to find a different, effective delivery means that can provide convenience to a user, which however is still inadequate.
Meanwhile, a body structure which is always in contact with an external environment, that is, skin, plays an important role as a protective barrier that prevents release of body fluids and infection, and water loss, and is composed of the epidermis, the dermis and subcutaneous tissue. The cornified layer of the epidermis is present at the outermost part of the skin, and prevents skin dryness by inhibiting the loss of water and electrolytes out of the skin and provides an environment facilitating normal biochemical metabolism of the skin. Also, the skin cornified layer plays an important role to protect the body from external physical damage and chemicals, and prevent dermal invasion by bacteria, molds, or viruses.
There are three absorption paths through the skin including absorption through the cornified layer, absorption through follicles and sebaceous glands, and absorption through sweat glands, and the delivery of active materials through the skin has numerous limitations in terms of the structural and physical characteristics of the skin. Particularly, the skin cornified layer has a compact structure at the outermost layer of the skin due to the natural death of keratinocytes, which are the main component cells of the skin, and exhibits an acidity of approximately pH 5 due to sweat and a variety of lipid ingredients. To pass through such a barrier of the cornified layer, it has been reported that the active material should generally have a molecular weight as small as 1,000 or less, and have lipophilic characteristics.
While low molecular weight synthetic compounds or natural materials which are frequently used as cosmetic and medical ingredients are known to be easily delivered into cells, since macromolecules such as proteins, peptides and nucleic acids are difficult to penetrate a cell membrane having a bilayer lipid membrane structure due to the size of a molecular weight and hydrophilicity, it has been known that, due to the intrinsic characteristics of the cornified layer that substantially constitutes the skin barrier, low molecular weight materials have extremely low penetration efficiency, and high molecular weight materials have an even lower penetration efficiency.
Therefore, for the transdermal delivery of botulinum toxin, a carrier which can deliver botulinum toxin through the skin barrier is needed. As a method of amplifying the efficiency of transmitting the small molecules and macromolecules through a cell plasma membrane, a protein transduction domain (PTD) may be applied. First, widely known PTDs are PTDs such as HIV-Tat, antennapedia, etc., which are known as positive-charged short peptides to deliver DNA, RNA, lipids, carbohydrates, compounds or viruses as well as proteins into cells. It has been reported that the PTDs are receptor-independent, and penetrate the cell membrane according to a mechanism such as endocytosis or phagocytosis. As a long history of such a PTD, a variety of applications using the PTD have been attempted, but it has been known that there is no successful development case so far. In the HIV-Tat-derived PTD, a peptide is derived from a virus, there is a problem in terms of safety, and particularly, when such transduction domains of the PTD family are independently used, it is known that an intracellular transduction rate is rapidly decreased at a low concentration of 2 to 5 μM or less according to the type of transduction domain. Also, it also has been reported that, when a protein having a molecular weight of 30,000 Da or more is conjugated to a PTD to be transduced into a cell, most of the PTD-protein conjugates tend to be transduced into the cell in the form of an endosome through endocytosis, and it has been reported that the endosome combines with a lysosome in the cytoplasm, and thus most of the PTD-protein conjugates are degraded by a hydrolase present in the lysosome, and only some undamaged PTD-protein conjugates are released into the cytoplasm. Accordingly, for dermal transduction of a functional protein using a PTD, a large amount of PTD-protein conjugates are needed to express expected efficacy, and will bring about an undesirable result in terms of economic feasibility.
To solve such a problem of the PTD and increase a pharmacological value, a hydrophobic or amphiphatic peptide having different characteristics from a conventional PTD, a macromolecule transduction domain (MTD; Korean Patent No. 10-1258279) was developed. An MTD is a novel cell-penetrating peptide, which has enhanced efficiency of delivering a material into cells, and has a different structure and different electrostatic properties, compared with a PTD. Unlike a PTD, in the intracellular transduction process of a MTD, endocytosis and energy are not needed, and the rigidity and integrity of the cell membrane act as important factors. Therefore, it has been suggested that direct interaction with the cell membrane is critical for the intracellular transduction process of an MTD. Such a cell membrane penetrating phenomenon of a peptide may increase a development value as a novel therapeutic drug by intracellular transduction of a therapeutic protein, or a nucleic acid material such as DNA or siRNA, which is difficult to be used as a drug because of a short in vivo half life or difficult cell membrane penetration. Also, compared with a conventional cell-penetrating peptide, which is a HIV-Tat-derived peptide, it is determined that a MTD has high availability in development of botulinum toxin as an external agent due to high efficiency of delivering a cargo material such as a compound, a peptide or a protein.
Also, as an amount of a light chain or light chain derivative of skin-penetrating and a nerve terminus cell-penetrating botulinum toxin, which is sought in the present invention, should be limited to a concentration of 1 to 10 ppm in order to ensure safety even through a toxicity attenuation process, it seems that it is inappropriate that a PTD is used as a skin- and neuronal cell-penetrating means, and to overcome this problem, there are demands for utilizing a MTD which has both skin barrier-penetrating and neuronal cell-penetrating potential and concentration-dependently penetrates the skin barrier even at low concentrations, or developing a novel MTD having the above-mentioned characteristics.